Epoxy ether type and isophthalene type saponin are the main saponins of Bupleurum chinense. However,due to the difference of their UV spectrum,there is no quantitative method for simultaneous determination of these two kinds of saponins. In this paper,a dual-wavelength high performance liquid chromatography(HPLC) was developed for simultaneous determination of five saponins in epoxidized ether(saikosaponin a,c,d) and isosorbide type(saikosaponin b1,b2). The mobile phase was eluted with acetonitrile-water(0.1% phosphoric acid) gradient at a column temperature of 30 °C and a flow rate of 1.0 mL·min⁻¹. The detection wavelengths were 208 nm for saikosaponins a,c, and d, and 254 nm for saikosaponins b₁ and b₂. The results showed that the separation of five kinds of saikosaponin was good, with the linear range of 9.70-1 935.00(=0.999 4),8.20-1 380.00(=0.999 3),6.90-1 640.00(=0.999 0),5.25-630.00(=0.999 4), and 5.15-618.00 mg·L⁻¹(=0.999 5), respectively. The average recoveries were 97.70%-100.2% and the RSD was less than 3%(=6). The method is simple,rapid and reproducible. It can be used for the determination of five kinds of saikosaponins in B. chinense.
Bupleurum ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Oleanolic Acid ; analogs & derivatives ; isolation & purification ; Plant Roots ; chemistry ; Saponins ; isolation & purification

5-Hydroxytryptamine 2C (5-HT) receptor is one of the major targets of anti-obesity agents, due to its role in regulation of appetite. In the present study, the 70% EtOH extract of the roots of Bupleurum chinense was revealed to have agonistic activity on 5-HT receptor, and the subsequent bioassay-guided isolation led to identification of several saikosaponins as the active constituents with 5-HT receptor agonistic activity in vitro and anti-obesity activity in vivo. The new compound, 22-oxosaikosaponin d (1), was determined by extensive spectroscopic analyses (HR-ESI-MS, IR, and 1D and 2D NMR). The primary structure-activity relationship study suggested that the intramolecular ether bond between C-13 and C-28 and the number of sugars at C-3 position were closely related to the 5-HT receptor agonistic activity. Saikosaponin a (3), the main saponin in B. chinense, showed obviously agonistic activity on 5-HT receptor with an EC value of 21.08 ± 0.33 μmol·Lin vitro and could reduce food intake by 39.1% and 69.2%, and weight gain by 13.6% and 16.4%, respectively, at 3.0 and 6.0 mg·kgin vivo. This investigation provided valuable information for the potential use of B. chinense as anti-obesity agent.
Animals ; Anti-Obesity Agents ; chemistry ; isolation & purification ; pharmacology ; Biological Assay ; Bupleurum ; chemistry ; Male ; Oleanolic Acid ; analogs & derivatives ; chemistry ; isolation & purification ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Saponins ; chemistry ; isolation & purification ; pharmacology ; Serotonin 5-HT2 Receptor Agonists ; chemistry ; isolation & purification ; pharmacology ; Structure-Activity Relationship

OBJECTIVEOleanolic acid (OA) has been reported to have anticancer effects, but the extent of its cytotoxicity, its ability to interact with nuclear DNA, its action against skin melanoma, as well as the molecular mechanism of its action against cell proliferation and in support of cell death are still unexplored. This led us to examine the efficacy of OA, a bioactive compound isolated from Phytolacca decandra, on these issues in the present investigation.

METHODSStudies related to analyses of cell viability, drug-DNA interaction, cell proliferation, cell cycle and epidermal growth factor receptor (EGFR) activity were performed. To investigate whether cells undergo apoptosis, studies like fluorescence microscopy, poly (ADP-ribose) polymerase (PARP) degradation, annexin V-fluorescein isothiocyanate/propidium iodide assay, alteration in mitochondrial membrane potential and activity of some relevant signaling proteins were performed.

RESULTSOA displayed a minimal and negligible cytotoxic effect on normal HaCaT cells (skin keratinocytes) and peripheral blood mononuclear cells but by contrast it reduced A375 cell viability significantly. OA interacted with nuclear DNA quickly after exposure. It acted as an anti-proliferative agent. It suppressed EGFR activity. OA administration led the cells to mitochondria-dependent caspase 3-mediated apoptosis.

CONCLUSIONOA interacts with cellular DNA, inhibits proliferation possibly through modulating EGFR activity and induces mitochondria-dependent caspase 3-mediated apoptosis in A375 cells which would qualify it as a potent anticancer agent.

Antineoplastic Agents ; isolation & purification ; therapeutic use ; Apoptosis ; drug effects ; Cell Line, Tumor ; DNA, Neoplasm ; drug effects ; Humans ; Melanoma ; drug therapy ; Microscopy, Fluorescence ; Oleanolic Acid ; isolation & purification ; therapeutic use ; Phytolacca ; chemistry ; Phytotherapy ; methods ; Plant Extracts ; isolation & purification ; therapeutic use ; Receptor, Epidermal Growth Factor ; drug effects ; physiology ; Signal Transduction ; drug effects ; Skin Neoplasms ; drug therapy

OBJECTIVETo determine the chemical constituents of Gentiana rhodantha.

METHODTo isolate the constituents, column chromatography over silica gel, MCI, Sephadex LH-20 and C18 reverse-phased silica gel were used. Spectroscopic methods were used to elucidate the structures of the isolated compounds.

RESULTSixteen compounds were isolated and elucidated as ten phonemic compounds, namely 1,3,7,8-tetrahydroxylxanthone (1), rhodanthenone D (2), 1,3,6,7-tetrahydroxylxanthone (3), 1,3,7-trihydroxy-4,8-dimethoxyxanthone (4), quercetin (5), isoorientin (6), mangiferin (7), norswertianolin (8), gallic acid ethyl ester (9) and salicylic acid (10), and six triterpenes including alpha-amyrin (11), erythrodiol 3-O-palmitate (12), ursolic aldehyde (13), uvaol 3-O-acetyl (14), ursolic acid (15) and 2alpha-hydroxyursolic acid (16).

CONCLUSIONCompounds 4-6, 8, 10-12, 15 and 16 were isolated from this plant for the first time. Compounds 1 and 3 were obtained firstly from the genus Gentiana and compounds 9, 13-14 were firstly from the family Gentianaceae.

Chromatography, Gel ; Chromatography, Reverse-Phase ; Dextrans ; Gentianaceae ; chemistry ; Mass Spectrometry ; Oleanolic Acid ; analogs & derivatives ; chemistry ; isolation & purification ; Plant Extracts ; chemistry ; isolation & purification ; Plants, Medicinal ; chemistry ; Quercetin ; chemistry ; isolation & purification ; Salicylic Acid ; chemistry ; isolation & purification ; Silica Gel ; Triterpenes ; chemistry ; isolation & purification ; Xanthones ; chemistry ; isolation & purification

OBJECTIVETo establish quality standard of Zhuang medicine Lonicera dasystyla, and provide scientific basis for the quality control of L. dasystyla.

METHODCharacteristics of materia medica, microscopic features, TLC indentification, inspection, extractum and determination of chlorogenic acid, macranthoidin B, dipsacoside B were carried out through the experience, microscopic, physical and chemical methods, respectively. The standard of quality control was formulated thereafter.

RESULTThe characteristics of materia medica, microscopic features, TLC indentification were specified, the average contents of water, total ash, acid-insoluble ash, alcohol-soluble extracts, chlorogenic acid were 11.6%, 6.6%, 0.2% , 24.4%, 1.16%, respectively, the total amount of macranthoidin B and dipsacoside B was 3.13%. Quality standard of L. dasystyla was proposed according to experimental results.

CONCLUSIONThe quality of L. dasystyla can be controlled effectively with the quality standard.

Chlorogenic Acid ; analysis ; isolation & purification ; Chromatography, Thin Layer ; Drugs, Chinese Herbal ; chemistry ; standards ; Lonicera ; chemistry ; Oleanolic Acid ; analogs & derivatives ; analysis ; isolation & purification ; Quality Control ; Saponins ; analysis ; isolation & purification ; Solubility

Based on previous report that the Chinese herb Ligustrum lucidum (LL) extract directly inhibited hepatitis C virus (HCV) replicase (NS5B) activity, the active components of LL extract to inhibit HCV NS5B activity and their inhibition mode were investigated in this study. LL extract was separated using ethyl acetate and thin layer chromatography (TLC). The inhibitory activity of separated fractions on HCV NS5B was analyzed by the inhibitory assay of NS5B activity. The results showed that only fractions 1 and 2 inhibited NS5B activity, and fraction 2 possessed higher inhibitory activity than fraction 1. HPLC analysis combined with inhibitory assays indicated that ursolic acid and oleanolic acid are the active components within fractions 1 and 2 to inhibit NS5B activity, separately. Moreover, oleanolic acid possessed higher inhibitory activity than ursolic acid. Further inhibition mode analysis found that both oleanolic acid and ursolic acid suppressed NS5B activity as noncompetitive inhibitors. The Ki values of ursolic acid and oleanolic acid were about 4.7 microg x mL(-1) (10 micromol x kg(-1)) and 2.5 microg x mL(-1) (5.5 micromol x kg(-1)), respectively. Taken together, these results demonstrated that oleanolic acid and ursolic acid suppressed NS5B activity as noncompetitive inhibitors, implying that the two natural products have potential value for HCV therapy.
Antiviral Agents ; isolation & purification ; pharmacology ; Chromatography, High Pressure Liquid ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Hep G2 Cells ; Humans ; Ligustrum ; chemistry ; Oleanolic Acid ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; Triterpenes ; isolation & purification ; pharmacology ; Viral Nonstructural Proteins ; antagonists & inhibitors ; metabolism

The ORF sequence of glycosyltransferase gene BcUGT1 cloned from Bupleurum chinense DC. was analyzed and its three dimentional structure was predicted. Using qRT-PCR method, the expression characteristics of BcUGT1 after methyl jasmonate (MeJA) induction and in different plant tissues were investigated. The results showed that BcUGT1 may be involved in saikosaponin biosynthesis in B. chinense. Thereafter, the recombinant vectors of BcUGT1 were constructed for its expression in E. coli. The target protein was successfully expressed and purified. In the present study, three vectors, pRSET-A, pET-28a (+) and pET-30a (+), and three isolates of E. coli, BL21 (DE3) plysS, BL21A1 and BL21-CodonPlus (DE3)-RIPL were used under different induction conditions, such as different concentrations and during times of inducers (L-arabinose and IPTG) and different inducing temperatures. The results showed that in the condition of 0.5 or 1 mmol x L(-1) IPTG, 16 degrees C, 20 h, target protein expressed in BL21-CodonPlus (DE3)-RIPL with pET-28a (+) or pET-30a (+) as vector. Using PrepEase His-tagged protein purification kit, the target protein was purified. The present work will be helpful for follow-up bio-function analysis of BcUGT1.
Amino Acid Sequence ; Base Sequence ; Bupleurum ; chemistry ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Plant ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Glycosyltransferases ; genetics ; isolation & purification ; metabolism ; Oleanolic Acid ; analogs & derivatives ; biosynthesis ; Open Reading Frames ; genetics ; Phylogeny ; Plants, Medicinal ; chemistry ; Protein Structure, Secondary ; Recombinant Fusion Proteins ; genetics ; metabolism ; Saponins ; biosynthesis

To study the chemical constituents of Dipsacus asper, chromatographic methods such as D101 macroporous resin, silica gel, octadecylsilyl (ODS) column chromatographic techniques and preparative HPLC were used, and five compounds were isolated from 70% (v/v) ethanol extract of the plant. By using spectroscopic techniques including 1H NMR, 13C NMR, 1H-1H COSY, HSQC, HMBC and TOF-MS, the compounds were identified as 3beta-hydroxy-24-nor-urs-4 (23), 12-dien-28-oic acid (1), ursolic acid (2), oleanolic acid (3), 3-O-alpha-L-rhamnosyl(1 --> 3)-beta-D-glucopyranosyl (1 --> 3)-alpha-L-rhamnosyl (1 --> 2)-alpha-L-arabinopyranosyl hederagenin 28-O-beta-D-glucopyranosyl (1 --> 6)-beta-D-glucopyranosyl ester (4), 3-O-[beta-D-xylopyranosyl (1 --> 4)-beta-D-glucopyranosyl (1 --> 4)] [alpha-L-rhamnosyl(1 --> 3)]-beta-D-glucopyranosyl (1 --> 3)-alpha-L-rhamnosyl(1 --> 2)-alpha-L-arabinopyranosyl hederagenin (5), separately. Among them, 1 is a new compound, and 2 is isolated from this plant for the first time.
Dipsacaceae ; chemistry ; Molecular Structure ; Oleanolic Acid ; chemistry ; isolation & purification ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Triterpenes ; chemistry ; isolation & purification

The roots of Pittosporum glabratum Lindl. (Pittosporaceae) have been used as a folk medicine for the treatment of rheumatic arthritis, insomnia and hypertension. Only a few chemical or biological studies on P. glabratum have been reported. As part of our ongoing phytochemical research on this plant, four compounds were isolated. Their structures were identified as 3beta, 6beta, 19alpha, 21alpha, 24-pentahydroxy-12-en-28-oleanolic acid (1), 3-O-beta-D-glucuronopyranosyl-28-O-beta-D-glucopyranosyl siaresinolic acid (2), 3, 4, 5-trimethoxyphenyl-1-O-beta-D-(5-O-syringoyl)-apiofuranosyl-(1 --> 6)-beta-D-glucopyranoside (3) and 3, 4, 5-trimethoxyphenol-1-O-beta-D-apiofuranosyl-(1 --> 6)-beta-D-glucopyranoside (4) on the basis of physical evidence and spectroscopic analysis. Among them, compound 1 is a new triterpenoid, and compounds 2-4 are isolated from the genus Pittosporum for the first time.
Molecular Structure ; Oleanolic Acid ; analogs & derivatives ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Rosales ; chemistry ; Triterpenes ; chemistry ; isolation & purification

OBJECTIVETo study the structures of secondary saikosaponins in saiko decoction.

METHODThe structures of saikosaponins were separated from saiko decoction and identified by various separation and spectral techniques. HPLC method was adopted for identifying all of separated saikosaponins.

RESULTEleven saikosaponins were separated from saiko decoction. Four of the seven new constitutes contained in saiko decoction were identified.

CONCLUSIONThis study lays a foundation for improving the identification of secondary saikosaponins.

Bupleurum ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Mass Spectrometry ; Molecular Structure ; Oleanolic Acid ; analogs & derivatives ; chemistry ; isolation & purification ; Saponins ; chemistry ; isolation & purification